Product Description
Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modeling of human colon mucus layers. Gut3Gel is presented here as a platform for culturing human microbiota samples. The protocol describes how to culture fecal samples in Gut3Gel using a multi-well plate format and how to prepare the samples for downstream analyses. Downstream assays include cell viability assessment, flow cytometry, metabolomics, and sequencing. The potential applications of Gut3Gel include microbial mining, drug screening, and permeability assays.
Components
2x multi-well plates containing gradient Gut3Gel colonic.
50 mL Bac3Gel® dissolution medium (50 mM sodium citrate).
Reagent Required But Not Provided
- 0.9% (w/v) NaCl solution prepared in deionized water.
Storage Conditions
Store in a dry place inside tightly sealed containers at 2-8 °C.
This product can be stored for at least 9 months.
Precautions and Disclaimer
For R&D use only.
Procedure
Before you begin
The protocol below describes the specific steps for using fecal samples. However, this protocol has also been used to culture monocultures, co-cultures, and microbial consortia. The Gut3Gel used in this protocol was produced in Brain Hearth Infusion (BHI) medium and is composed of 50 mg.mL-1 porcine stomach type III mucin. Gut3Gel’s composition is fully tunable; customized compositions are commercially available (i.e. incorporation of other mucin types, proteins, lipids and media). The cross-linking and oxygen gradients within Gut3Gel are intrinsic features established during the production process. These are dynamically affected by microbial culturing.
Ensure all instruments and consumables (blender, colander, pipettes, falcons, and microcentrifuge tubes) are sterilized and free of contaminants.
All manipulations should be performed under aseptic conditions, ideally in a Class II biological safety cabinet.
Prepare a 0.9% (w/v) sterile NaCl solution.
A. Fecal Sample Processing
1. Weigh at least 30 g of fresh fecal sample and transfer to a sterilized blender.
For each 30 g of fecal sample, add 150 mL of sterile 0.9% NaCl solution.
Homogenize the mixture until a uniform slurry is obtained.
2. To remove large particulate matter, filter the slurry through a sterilized colander into a sterile container.
Aliquot into 50 mL falcon tubes.
Keep the filtered slurry on ice until use (do not exceed 1 hour).
Filtered slurry can be aliquoted, supplemented with glycerol (final concentration of 10% v/v) and stored at -80 °C for future experiments. This should be done as quickly as possible to preserve microbial diversity.
Fecal samples should be as fresh as possible; ideally, processing should start within 30 minutes from defecation time.
Rapid processing is crucial to maintain microbial viability.
Avoid excessive blending to prevent mechanical damage to microbes.
B. Culturing in Gut3Gel
1. Prepare Gut3Gel multi-well plates at room temperature. For this protocol, 24-well plates are used as example.
2. Add fecal slurry to each well in a 1:1 volume ratio with Gut3Gel.
Example: For a 24-well plate, add 700 μL of fecal slurry as each well contains 700 μL of Gut3Gel.
Volumes for other plate formats:
- 6-well: 3,700 μL
- 12-well: 1,600 μL
- 48-well: 350 μL
- 96-well: 100 μL
3. Cover the plates and incubate at 37 °C under aerobic or anaerobic conditions for up to 72 hours depending on experimental design.
The recommended incubation period is 72 hours but it may be adjusted as required by the user.
To test the effects of molecules (e.g. biotics, active principles) on microbiota, it is recommended to let microbiota stabilize in Gut3Gel for 24 hours at 37 °C before adding the molecule of interest.
The multi-well plates can be sealed with a breathable sealing membrane prior to incubation to prevent cross-contamination.
The fecal slurry should be added gently (to avoid cross-contamination) to the top of Gut3Gel. Do not mix.
While not a strict requirement, ensuring anaerobic conditions prior culturing in Gut3Gel will prevent the loss of obligate anaerobes.
C. Dissolution of Gut3Gel
1. For each well, remove the supernatant from the surface of Gut3Gel by gentle aspiration, without disturbing the gel.
Wash the gels by adding 700 μL (for 24-well plates) of 0.9% NaCl solution.
Gently aspirate and discard the supernatant.
The washing step ensures that we only consider microbes colonizing Gut3Gel.
2. Add Bac3Gel® dissolution medium (50 mM sodium citrate solution) to each well. Mix thoroughly to fully dissolve Gut3Gel structure.
- To calculate the volume of Bac3Gel® dissolution medium to add to each well, enter the volume (μL) of Gut3Gel according to the multi-well plate typology and click Calculate:
- Transfer the homogenized samples to microcentrifuge tubes.
Full dissolution of Gut3Gel can take some time depending on the plate format and on the presence of exopolysaccharides.
For sequencing: Centrifuge the samples (14,000 x g, 10 minutes), then use the pellet for DNA extraction.
For metabolomics: Centrifuge the samples (14,000 x g, 10 minutes), then collect and lyophilize the supernatant.
Other applications include assessing cell viability, performing flow cytometry analysis, measuring metabolic activity, and conducting standard microbiological characterization assays.
Troubleshooting
Problem 1
The gels are not dissolving properly or take too long to dissolve.
Potential solution
Solution 1: Increase the volume of 50 mM sodium citrate and mix.